Structural studies on bovine rhodopsin are to be completed and various functional groups in the sequence identified. The cyanogen bromide peptides will be isolated and characterized. They will be placed in order using methionine sequences already determined. The cyanogen bromide peptides will help in ordering the smaller peptides we have already sequenced and will provide the few segments previously not accounted for. Cysteine residues in the rhodopsin sequence will be identified as C14-carboxymethylcysteine, and any cystine residues as aminoethylcysteine. Reactivity of rhodopsin cysteine residues in the rod outer segment membranes will be determined toward a variety of sulfhydryl reagents both in the native and bleached membranes. The functional effect of such modifications on membrane osmotic properties and rhodopsin regenerability will be assessed. Time course of the in vitro phosphorylation of rhodopsin will be studied and the specificity of the reaction will be determined by peptide analysis. The site in the sequence for the early introduction of 32p-phosphate will be located. The location of rhodopsin in disk membranes will be investigated. Cross-linking agents will be used to detect differences in location as a function of bleaching. Proteolytic digestion will be employed as a probe to detect what segments of the rhodopsin sequence are surface exposed and how this exposure changes upon bleaching. The functional as well as structural consequences of limited proteolysis on disk membranes will be studied.